Aims/hypothesis. Glucagon-like peptide-1 ameliorates the symptoms of diabetes through stimulation of insulin secretion and enhancement of beta-cell mass. We have therefore investigated the effects of glucagonlike peptide-1 on the development of diabetes, using db/db mice as a model of Type II diabetes. Methods. The potent glucagon-like peptide-1 analogue Exendin-4 or vehicle (control) was administered (i.p.; 1 nmol/kg) to obese 6-week old db/db mice daily for 14 days (n=10). Results. By 8 weeks of age, control db/db mice developed hyperglycaemia (fasting: 10.4±0.5 mmol/l), hyperinsulinaemia and impaired glucose tolerance. However, Exendin-4 treatment prevented hyperglycaemia (fasting: 6.1±1.0 mmol/l, p<0.01), with reduced plasma insulin concentrations (p<0.001) and improved glucose tolerance (p<0.05). Peripheral insulin sensitivity was not affected. However, insulin release in vivo and in vitro from the perfused pancreas was improved by Exendin-4, as were pancreatic insulin concentrations (0.54±0.02 vs 0.32±0.01 µg/mg protein, p<0.05). These changes occurred in conjunction with increased beta-cell mass (3.01±0.31 vs 2.22±0.22 mg, p<0.05) and proliferation (BrdU + beta-cells: 1.08±0.20 vs 0.47±0.11%, p<0.05), as well as decreased apoptosis (Tunel + beta-cells: 0.37±0.06 vs 1.20±0.21%). Western blot demonstrated increased expression of Akt1 (by fivefold, p<0.01) and p44 MAP kinase (by sixfold, p<0.01), and decreased activation of caspase-3 (by 30%, p<0.05). Conclusion/interpretation. Our results suggest that Ex4 treatment delays the onset of diabetes in 6-8 week old db/db mice, through a mechanism involving Akt1 and expansion of the functional beta-cell mass.

Aims/hypothesis. The incretin hormone glucagon-like peptide-1 augments islet cell mass in vivo by increasing proliferation and decreasing apoptosis of the beta cells. However, the signalling pathways that mediate these effects are mostly unknown. Using a clonal rat pancreatic beta cell line (INS-1), we examined the role of protein kinase B in mediating beta-cell growth and survival stimulated by glucagon-like peptide-1. Methods. Immunoblot analysis was used to detect active (phospho-) and total protein kinase B. Proliferation was assessed using 3 H-thymidine incorporation, while apoptosis was quantitated using 4′-6-diamidino-2-phenylindole staining and APO percentage apoptosis assay. Kinase-dead and wild-type protein kinase B was introduced into cells using adenoviral vectors. Results. Glucagon-like peptide-1 rapidly activated protein kinase B in INS-1 cells (by 2.7±0.7-fold, p<0.05). This effect was completely abrogated by inhibition, with wortmannin, of the upstream activator of protein kinase B, phosphatidylinositol-3-kinase. Glucagon-like peptide-1 also stimulated INS-1 cell proliferation in a dose-dependent manner (by 1.8±0.5-fold at 10 −7 mol/l, p<0.01), and inhibited staurosporine-induced apoptosis (by 69±12%, p<0.05). Both of these effects were also prevented by wortmannin treatment. Ablation of protein kinase B by adenovirus-mediated overexpression of the kinase-dead form of protein kinase Bα prevented protein kinase B phosphorylation and completely abrogated both cellular proliferation (p<0.05) and protection from drug-induced cellular death (p<0.01) induced by glucagon-like peptide-1. Conclusions/interpretation. These results identify protein kinase B as an essential mediator linking the glucagon-like peptide-1 signal to the intracellular machinery that modulates beta-cell growth and survival. [Diabetologia (2004) 47:478-487]

Glucagon-like peptide (GLP-1), a major physiological incretin, plays numerous important roles in modulating blood glucose homeostasis and has been proposed for the treatment of type 2 diabetes. The major obstacles for using native GLP-1 as a therapeutic agent are that it must be delivered by a parenteral route and has a short half-life. In an attempt to develop a strategy to prolong the physiological t 1/2 and enhance the potency of GLP-1, a fusion protein consisting of active human GLP-1 and mouse IgG 1 heavy chain constant regions (GLP-1/Fc) was generated. A plasmid encoding an IgK leader peptide-driven secretable fusion protein of the active GLP-1 and IgG 1 -Fc was constructed for mammalian expression. This plasmid allows for expression of bivalent GLP-1 peptide ligands as a result of IgG-Fc homodimerization. In vitro studies employing purified GLP-1/ Fc indicate that the fusion protein is functional and elevates cAMP levels in insulin-secreting INS-1 cells. In addition, it stimulates insulin secretion in a glucose concentrationdependent manner. Intramuscular gene transfer of the plasmid in db/db mice demonstrated that expression of the GLP-1/Fc peptide normalizes glucose tolerance by enhancing insulin secretion and suppressing glucagon release. This strategy of using a bivalent GLP-1/Fc fusion protein as a therapeutic agent is a novel approach for the treatment of diabetes.